von Willebrand factor (VWF) is a pro-coagulant glycoprotein expressed only in endothelial
cells and megakaryocytes. It plays a critical role in hemostasis and thrombus formation.
It mediates the adhesion of platelets to the endothelium/sub-endothelium surfaces,
which is the primary step in thrombogenesis. Our previous analyses demonstrated that
hypoxia induced an increase in levels, and alteration in vascular tree expression
pattern, of the VWF specifically in the lungs of mice. Alteration in expression pattern
corresponded to a significant increase in the proportion of lung microvascular endothelial
cells that exhibited de novo activation of VWF expression. Hypoxia-induced elevated
and altered VWF expression was correlated with a significant increase in platelet
aggregates formation in the lung vasculature. Since during organ-transplantation donor
organs are under hypoxic conditions, we explored whether this may lead to alterations
in VWF expression and whether modification of transplantation procedure to reduce
hypoxic exposure could prevent such alterations.
METHODS AND RESULTS
Procured pig's lungs that were maintained in static cold storage “SCS” or exposed
to ex vivo lung perfusion (before and after perfusion) were used. Lung tissue biopsies
were obtained immediately after organ harvest, 12 hours post cold storage, or post
warm perfusion. VWF RNA and protein expression levels were analyzed using RT-PCR and
western blot. In addition, we proceeded to determine whether SCS preservation also
alters the proportion of pig's lung vascular endothelial cells that exhibit VWF expression.
For these analyses, we performed double stained immunofluorescence (IF) analyses using
CD31 (endothelial cell marker) and VWF antibodies on samples of lungs that were preserved
and transplanted. Results demonstrated that VWF mRNA and protein levels were similar
after 12 hours of cold storage compared to immediate harvest, but were significantly
reduced in lungs that were perfused under ex vivo lung perfusion “EVLP” for 12 hours.
Furthermore, IF analysis demonstrated that preservation of lungs under cold storage
conditions prior to transplantation altered VWF expression pattern leading to an increasing
number of microvascular (indicated by CD31 staining) endothelial cells that express
Increased VWF expression in microvascular endothelial cells under SCS may contribute
to transplant associated thrombogenicity. Reduction of VWF expression through ex vivo
normothermic perfusion may have a significant effect in reducing potential thrombogenic
consequences, which is a major complication of organ transplantation.